This project will focus on chondroitin sulfate glycosaminoglycans (CS GAGs), a class of polysaccharides that play important roles in development, viral invasion, cancer, and spinal cord injury. CS GAGs display diverse sulfation patterns that are spatiotemporally regulated in vivo. However, efforts to identify functions for specific sulfation motifs have been hampered by the structural complexity of CS and a lack of tools. In this grant, we will combine the power of both organic chemistry and biology to overcome these challenges and identify novel functions for specific motifs in the nervous system. The broad objectives of this program are to: (1) advance a fundamental understanding of the structure-function relationships of CS GAGs, (2) understand the roles of CS GAGs in neuroplasticity and regeneration, and (3) develop new approaches to study and manipulate GAG-mediated biological processes, with the long-term goal of stimulating synaptic plasticity and neuronal repair. In the last granting period, we developed a set of chemical tools to study specific sulfation motifs and discovered that a particular motif, CS-E, inhibits axon regeneration after spinal cord injury. Blocking the CS-E motif using an anti-CS-E antibody stimulated axon regeneration in vivo. Moreover, we found that this same motif repels axons and plays a critical role in neural circuit formation during brain development. An important observation from this work was that the activity of CS-E required its interaction with cell-surface receptors and activation of specific inhibitory signaling pathways in neurons. In the present grant, we will develop new approaches to modulate the interactions of CS-E with neuronal receptors, including the viral-mediated delivery of single-chain anti-CS-E antibodies, small- molecule sulfotransferase inhibitors, and glycopolymer mimetics (Aim 1). We will study how CS-E regulates protein signaling complexes, with a particular focus on semaphorin-3A/neuropilin-1/plexin A (Sema3A/Nrp1/PlxnA) and ephrin/Eph receptor (Efn/Eph) complexes (Aims 2a and 3a). Finally, we will investigate the ability of the agents developed in Aim 1 to promote neuroplasticity in the visual cortex (Aims 2b,c) and axon regeneration after spinal cord injury (Aim 3b). These studies are expected to provide new chemical tools to advance an understanding of GAGs and fundamentally change how CS GAGs are viewed - from being static, passive molecules to ligands that actively regulate important signaling pathways. Finally, if successful, the agents developed in Aim 1 could lead to novel therapeutic strategies for stimulating neuronal plasticity and repair in the case of aging, injury, and disease.